PROJECT SUMMARY/ABSTRACT Ultrasound is among the world's most widely used biomedical imaging technologies due to its relative simplicity, low cost and ability to visualize deep tissues with high spatial and temporal resolution. However, ultrasound has historically had a small role in molecular and cellular imaging due to the lack of contrast agents connected to specific aspects of cellular function such as gene expression. To address this limitation, we are developing the first acoustic biomolecules ? proteins that can be imaged with ultrasound. These constructs are based on gas vesicles ? a unique class of gas-filled proteins from buoyant photosynthetic microbes, which we adapted as imaging agents for ultrasound in 2014. Since this key initial discovery, our laboratory has led the development of the emerging field of biomolecular ultrasound by engineering the physical, chemical and biological properties of gas vesicles to enable multiplexed imaging, cellular targeting and selective detection in vivo. In parallel, we have worked on transplanting the genetic program encoding gas vesicles into heterologous hosts, recently succeeding in doing so in commensal bacteria relevant to the mammalian microbiome, while in parallel making initial progress on expressing gas vesicles in mammalian cells. In addition, we discovered that gas vesicles can produce susceptibility-weighted MRI contrast erasable by ultrasound, providing an additional readout modality with unique advantages. Here we propose to build on these insights to advance gas vesicles as targeted nanoscale contrast agents, mammalian reporter genes and functional sensors for ultrasound. This work will focus on engineering gas vesicle properties for long-term circulation and extravascular targeting through the bloodstream, achieving robust expression of gas vesicles as reporter genes in mammalian cells, developing nonlinear ultrasound pulse sequences to maximize the sensitivity of gas vesicle imaging, and designing the first acoustic sensors of enzyme activity. The fundamental innovation contained in this research is that gas vesicle are the first biomolecular, genetically engineered and encoded contrast agent of any kind for ultrasound. As a result, they have the potential to transform this imaging modality analogously to the way fluorescent proteins transformed optical microscopy.